This invention relates to chromatographic separation techniques and to materials useful in such techniques. More particularly, it relates to the chromatographic use of semipermeable microcapsules.
Chromatography procedures are used to separate the components of a mixture of solutes. Basically, they involve the flow of a mobile phase of gas or liquid containing a mixture of solutes over a stationary phase. As the mobile phase moves past the stationary phase, repeated sorption and desorption of the solutes occur at rates determined, among other factors, by each solute's ratio of distribution between the phases. The solutes in the mixture thus move at different rates along the chromatographic flow path, producing a characteristic pattern of zones rich in individual solutes called a chromatogram. Usually, the separate fractions are eluted from the flow path for identification or other purposes.
Zeolites, and certain cross-linked polysaccharides, polystyrenes, polyacrylamides, and agarose gels are employed in a type of chromatography known as gel filtration. These substances are often referred to as "molecular sieves". All are characterized by an open structure through which solutes of various critical diameter pass at different rates. When packed in a column, these materials effectively separate the components of a mixture of solutes on the basis of their critical diameter. Prior to use, these materials must be swelled by immersion in a solvent, usually water, for a time sufficient to establish equilibrium and to stabilize pore size. Cross-linked polyacrylamide and polysaccharide gels are capable of resolving solutes over a wide molecular weight range, up to about 200,000 for polysaccharides, and up to about 800,000 for proteins. Agarose gels separate mixtures containing molecules in a molecular weight range of approximately 0.5.times.10.sup.5 to 150.times.10.sup.6. Zeolites separate materials in the molecular weight range below about 200 daltons.
The cross-linked polymeric materials discussed above are only marginally effective as molecular sieves for separating substances of molecular weight in the range below about 1000-2000 daltons, and the zeolites are unsuitable for resolving solutes of molecular weight above about 200 daltons.
Recently, another separation technique known as affinity chromatography has been developed. Affinity chromatography involves immobilizing on a suitable support one component of a two-component specific binding complementary pair such as the antibody of an antibody-antigen pair or the hormone binding protein of a binding protein-hormone pair. The immobilized component is then packed into a column. On passage of a mixture of materials, one component of which comprises the immobilized substance's complementary binding components, the complementary component is selectively extracted. The resulting bond can be broken with a suitable reagent such as an acid solution or a solution of high salt concentration, and the concentrated component can be recovered. This technique has found utility in various research efforts and in the purification of complex proteins.
In a related technique enzyme is immobilized on a substrate and packed into a column for use as a reusable enzyme reactor. Solutions containing the enzyme's substrate or substrate system may then be passed through the system and chemically changed by the catalytic influence of the enzyme.